期刊
CATALYSIS SCIENCE & TECHNOLOGY
卷 1, 期 6, 页码 948-957出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/c0cy00092b
关键词
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资金
- UK Biotechnology and Biological Sciences Research Council (BBSRC)
- Royal Society
- BBSRC [BB/D01963X/1, BB/G023581/1, BB/G023581/2, BB/D002826/1, BB/E010717/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D002826/1, BB/G023581/1, BB/D01963X/1, BB/G023581/2, BB/E010717/1] Funding Source: researchfish
This work describes a site-directed mutagenesis study of pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of key active site residues in influencing both product enantiopurity and the ratio of C=C vs. nitro-group reduction with 2-phenyl-1-nitropropene. Comparative biotransformations of wild type and single/double mutants of PETN reductase with 2-phenyl-1-nitropropene showed that one enzyme scaffold was capable of generating both enantiomeric products with improved enantiopurities by a manipulation of the reaction conditions and/or the presence of a one or two key mutations. These changes located at key active site residues were sufficient to moderately improve product enantiopurity, cause a switch in the major product enantiomer formed and/or promote or eliminate side-product formation. The mutation of substrate-binding residue Y351 to alanine and phenylalanine improved the biocatalytic potential of PETN reductase by the elimination of a competing side reaction. The crystal structures of three mutants at residue Y351 (PDB codes: 3P81, 3P84 and 3P8J) show that only subtle changes in the active site environment may be necessary to generate significantly improved biocatalysts.
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